Methods for controlled proliferation of stem cells / generating inner ear hair cells using 1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl based compounds

ABSTRACT

The present invention relates to methods of inducing the self-renewal of stem/progenitor supporting cells, including inducing the stem/progenitor cells to proliferate while maintaining, in the daughter cells, the capacity to differentiate into hair cells. Specifically, the invention relates to methods of using compounds comprising a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indole moiety having a Formula I 
                         
and pharmaceutically acceptable salts thereof.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalapplication No. 62/302,753, filed Mar. 2, 2016, the entire contents ofwhich are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of using1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indole based compoundsinducing the self-renewal of stem/progenitor supporting cells, includinginducing the stem/progenitor cells to proliferate while maintaining, inthe daughter cells, the capacity to differentiate into tissue cells.

BACKGROUND OF THE INVENTION

Stem cells exhibit an extraordinary ability to generate multiple celltypes in the body. Besides embryonic stem cells, tissue specific stemcells serve a critical role during development as well as in homeostasisand injury repair in the adult. Stem cells renew themselves throughproliferation as well as generate tissue specific cell types throughdifferentiation. The characteristics of different stem cells vary fromtissue to tissue, and are determined by their intrinsic genetic andepigenetic status. However, the balance between self-renewal anddifferentiation of different stem cells are all stringently controlled.Uncontrolled self-renewal may lead to overgrowth of stem cells andpossibly tumor formation, while uncontrolled differentiation may exhaustthe stem cell pool, leading to an impaired ability to sustain tissuehomeostasis. Thus, stem cells continuously sense their environment andappropriately respond with proliferation, differentiation or apoptosis.It would be desirable to drive regeneration by controlling the timingand extent of stem cell proliferation and differentiation. Controllingthe proliferation with small molecules that are cleared over time wouldallow for control of the timing and extent of stem cell proliferationand differentiation. Remarkably, tissue stem cells from differenttissues share a limited number of signaling pathways for the regulationof their self-renewal and differentiation, albeit in a very contextdependent manner. Some of these pathways are the Wnt, GSK3-alpha, andGSK3-beta pathways.

Lgr5 is expressed across a diverse range of tissues and has beenidentified as a biomarker of adult stem cells in a variety of tissuessuch as the gut epithelia (Barker et al. 2007), kidney, hair follicle,and stomach (Barker et al, 2010; Haegebarth & Clevers, 2009). Forexample, it was first published in 2011, that mammalian inner ear haircells are derived from LGR5⁺ cells (Chai et al, 2011, Shi et al. 2012).Lgr5 is a known component of the Wnt/beta-catenin pathway, which hasbeen shown to play major roles in differentiation, proliferation, andinducing stem cell characteristics (Barker et al. 2007).

Permanent damage to the hair cells of the inner ear results insensorineural hearing loss, leading to communication difficulties in alarge percentage of the population. Hair cells are the receptor cellsthat transduce the acoustic stimulus. Regeneration of damaged hair cellswould provide an avenue for the treatment of a condition that currentlyhas no therapies other than prosthetic devices. Although hair cells donot regenerate in the mammalian cochlea, new hair cells in lowervertebrates are generated from epithelial cells, called supportingcells, that surround hair cells.

Prior work has focused on transdifferentiation of supporting cells intohair cells through activation or forced expression of genes that lead tohair cell formation, with a particular focus on mechanisms to enhanceexpression of Atoh1 (Bermingham et al., 1999; Zheng and Gao, 2000;Izumikawa et al., 2005; Mizutari et al., 2013). Interestingly, cellstransduced with Atoh1 vectors have been shown to acquire vestibularphenotypes (Kawamoto et al., 2003; Huang et al., 2009; Yang et al.,2012, 2013), and lack complete development. As mentioned, upregulatingAtoh1 via gene insertion has been shown to create non-cochlear celltypes that behave in a manner that is not found within the nativecochlea. In addition, these methods increase hair cell numbers butdecrease supporting cell numbers. Since supporting cells are known tohave specialized roles (Ramirez-Camancho 2006, Dale and Jagger 2010),loss of these cells could create problems in proper cochlear function.

Thus, there remains a long felt need to protect auditory cells beforeinjury and preserve/promote the function of existing cells after injury.There remains a need to regenerate cochlear supporting cells or haircells after injury. As disclosed below, in certain embodiments, thepresent invention provides methods for preventing and treating auditorydysfunctions.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method forproliferation of stem cells comprising administering to a cellpopulation an effective amount of a compound or composition, orpharmaceutically acceptable salts thereof, provided herein. In someembodiments, proliferation occurs in the absence of a notch activator oran HDAC inhibitor.

Among the various aspects of the present disclosure, therefore, may benoted a method for activating the Wnt pathway in a cell population toincrease the capacity of the population for self-renewal, i.e., thecapacity for repeated generation of daughter cells with equivalentproliferation and ‘cell fate specification’ potential, anddifferentiation, i.e., the capacity for generation of daughter cellsspecified for differentiation. In one embodiment, the cell population isa cochlear supporting cell population. Preferably, the Wnt pathway isactivated upstream of the c-myc gene in members of the population andwithout any genetic modification of the population. Instead, the Wntpathway is preferably activated by small molecules that transientlyinduce such activity. Additionally, the supporting cell populationpreferably includes supporting cells that are LGR5⁺ and endogenous tothe Organ of Corti.

A further aspect of the present disclosure is a method for inducing theself-renewal of stem/progenitor supporting cells comprised by a cochlearcell population. That is, the stem/progenitor supporting cells areinduced to proliferate (i.e., divide and form daughter cells) whilemaintaining, in the daughter cells, the capacity to differentiate intohair cells. In contrast, if the stem/progenitor supporting cells weremerely induced to proliferate (without maintaining multi-potency), thedaughter cells would lack the capacity to divide into hair cells.Further, merely enforcing differentiation of a pre-existingstem/progenitor cell population has the potential to exhaust the stemcell pool. Proliferation is preferably activated by small molecules thattransiently induce such activity. Additionally, in certain embodimentsthe supporting cell population preferably includes supporting cells thatare LGR5+ and endogenous to the Organ of Corti.

In a first aspect, the present disclosure provides methods of using1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indole containing compounds,or pharmaceutically acceptable salts thereof, for inducing theself-renewal of stem/progenitor supporting cells is provided. The1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl based compounds, orpharmaceutically acceptable salts thereof, comprising the followingstructural moiety of Formula I within the compound:

In some embodiments, the 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indolyl based compound is a3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound, or pharmaceutically acceptable salts thereof. The3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compounds comprising the following structural moiety ofFormula II within the compound:

In some embodiments, the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione,or pharmaceutically acceptable salts thereof, having a Formula III:

which is disclosed in U.S. Pat. No. 8,318,713, herein incorporated byreference in its entirety.

In certain embodiments, therefore, the present disclosure providesmethods to induce self-renewal of a population of supporting cells byactivating pathways and mechanisms that are known to be involved ininducing stem cell properties, such as those used to create “inducedpluripotent stem cells”. Preferably, the pathways are activated withsmall molecules. For example, a compound when applied in vitro to asupporting cell population induces the population to proliferate to ahigh degree and in high purity in a Stem Cell Proliferation Assay, andalso allows the population to differentiate into a high puritypopulation of a tissue cell in a Stem Cell Differentiation Assay. In onesuch embodiment, the compound induces and maintains stem cell propertiesby proliferating to produce stem cells that can divide for manygenerations and maintain the ability to have a high proportion of theresulting cells differentiate into tissue cells. Further, theproliferating stem cells express stem cell markers which may include oneor more of Lgr5, Sox2, Opem1, Phex, lin28, Lgr6, cyclin D1, Msx1, Myb,Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog, Esrrb, Rex1, Dnmt3a,Dnmt3b, Dnmt31, Utf1, Tel1, Oct4, Klf4, Pax6, Six2, Zic1, Zic2, Otx2,Bmi1, CDX2, STAT3, Smad1, Smad2, smad2/3, smad4, smad5, and smad7.

In certain embodiments, the disclosure provides a method for expanding apopulation of cochlear cells in a cochlear tissue comprising a parentpopulation of cells. In this embodiment, the method comprises contactingthe cochlear tissue with a stem cell proliferator to form an expandedpopulation of cells in the cochlear tissue, wherein:

-   -   the stem cell proliferator is capable of (i) forming a        proliferation assay final cell population from a proliferation        assay initial cell population over a proliferation assay time        period in a stem cell proliferation assay and (ii) forming a        differentiation assay final cell population from a        differentiation assay initial cell population over a        differentiation assay time period in a stem cell differentiation        assay wherein:    -   (a) the proliferation assay initial cell population has (i) a        proliferation assay initial number of total cells, (ii) a        proliferation assay initial number of Lgr5⁺ cells, (iii) a        proliferation assay initial number of hair cells, (iv) a        proliferation assay initial Lgr5⁺ cell fraction that equals the        ratio of the proliferation assay initial number of Lgr5⁺ cells        to the proliferation assay initial number of total cells,        and (v) a proliferation assay initial hair cell fraction that        equals the ratio of the proliferation assay initial number of        hair cells to the proliferation assay initial number of total        cells;    -   (b) the proliferation assay final cell population has (i) a        proliferation assay final number of total cells, (ii) a        proliferation assay final number of Lgr5⁺ cells, (iii) a        proliferation assay final number of hair cells, (iv) a        proliferation assay final Lgr5⁺ cell fraction that equals the        ratio of the proliferation assay final number of Lgr5⁺ cells to        the proliferation assay final number of total cells and (v) a        proliferation assay final hair cell fraction that equals the        ratio of the proliferation assay final number of hair cells to        the proliferation assay final number of total cells;    -   (c) the differentiation assay initial cell population has (i) a        differentiation assay initial number of total cells, (ii) a        differentiation assay initial number of Lgr5⁺ cells, (iii) a        differentiation assay initial number of hair cells, (iv) a        differentiation assay initial Lgr5⁺ cell fraction that equals        the ratio of the differentiation assay initial number of Lgr5⁺        cells to the differentiation assay initial number of total        cells, and (v) a differentiation assay initial hair cell        fraction that equals the ratio of the differentiation assay        initial number of hair cells to the differentiation assay        initial number of total cells;    -   (d) the differentiation assay final cell population has (i) a        differentiation assay final number of total cells, (ii) a        differentiation assay final number of Lgr5⁺ cells, (iii) a        differentiation assay final number of hair cells, (iv) a        differentiation assay final Lgr5⁺ cell fraction that equals the        ratio of the differentiation assay final number of Lgr5⁺ cells        to the differentiation assay final number of total cells,        and (v) a differentiation assay final hair cell fraction that        equals the ratio of the differentiation assay final number of        hair cells to the differentiation assay final number of total        cells;    -   (e) the proliferation assay final number of Lgr5⁺ cells exceeds        the proliferation assay initial number of Lgr5⁺ cells by a        factor of at least 10; and    -   (f) the differentiation assay final number of hair cells is a        non-zero number.

The assay described above does not include applying a notch activator oran HDAC inhibitor.

In certain embodiments, the disclosure provides a method for increasingthe cell density of supporting cells in a population of cochlear cells.The method comprises activating pathways and mechanisms that induce stemcell properties in the supporting cells, proliferating the activatedsupporting cells (while maintaining the multi-potent character of thesupporting cells in the newly formed daughter cells) and thereafterallowing (or even inducing) the expanded population to differentiateinto hair cells to form an expanded cochlear cell population wherein thecell density of hair cells in the expanded cochlear cell populationexceeds the cell density of hair cells in the original (non-expanded)cochlear cell population. In some embodiments, such proliferation occursin the absence of a notch activator or an HDAC inhibitor. In someembodiments, the supporting cell population is an in vitro supportingcell population. In other embodiments, the supporting cell population isan in vivo supporting cell population. Additionally, the proliferationstage is preferably controlled to substantially maintain the nativeorganization of the cochlear structure. The proliferation is induced bythe compound described herein that transiently induces such activityrather than by induction of c-myc and without any genetic modificationof the population. In some embodiments, such proliferation occurs in theabsence of a notch activator or an HDAC inhibitor. Additionally, incertain embodiments the supporting cell population preferably includessupporting cells that are LGR5⁺ and endogenous to the Organ of Corti.

In certain embodiments, the disclosure provides a method for increasingthe cell density of Lgr5⁺ supporting cells in a population of cochlearcells. The method comprises activating pathways and mechanisms thatinduce or maintain stem cell properties in the Lgr5⁺ supporting cells,proliferating the activated Lgr5⁺ supporting cells (while maintainingsuch stem cell properties) and thereafter allowing (or even inducing)the expanded population to differentiate into hair cells to form anexpanded cochlear cell population wherein the cell density of hair cellsin the expanded cochlear cell population exceeds the cell density ofhair cells in the original (non-expanded) cochlear cell population. Insome embodiments for increasing the cell density of Lgr5⁺ supportingcells in a population of cochlear cells, such increasing of the celldensity occurs in the absence of a notch activator or an HDAC inhibitor.In some embodiments, the Lgr5⁺ supporting cell population is an in vitroLgr5⁺ stem cell population. In other embodiments, the Lgr5⁺ supportingcell population is an in vivo supporting cell population. Additionally,in certain embodiments the proliferation stage is preferably controlledto substantially maintain the native organization of the cochlearstructure.

In certain embodiments, the disclosure provides a method for increasingthe cell density of hair cells in an initial population of cochlearcells, the initial population (which may be an in vivo or an in vitropopulation) comprises hair cells, Lgr⁻ supporting cells, and Lgr5⁺supporting cells. In some embodiments for increasing the cell density ofhair cells in an initial population of cochlear cells, such increasingof the cell density occurs in the absence of a notch activator or anHDAC inhibitor. The method comprises administering to the initialpopulation a compound described herein.

In certain embodiments, the method produces stem cells in a Stem CellProliferation Assay that express stem cells markers Lgr5⁺. In certainembodiments, if a mixed population of Lgr5⁺ and non-Lgr5⁺ stems areplaced in a Stem Cell Proliferation Assay, the method increases thefraction of cells in the population that are Lgr5⁺. In some embodiments,such production of stem cells in a Stem Cell Proliferation Assay occursin the absence of a notch activator or an HDAC inhibitor.

Expanding supporting cell populations to a degree that destroys thenative organization of the cochlear structure could inhibit cochlearfunction. Driving proliferation of existing supporting cells with asmall molecule signal may allow for a more controlled regeneration ofhair cells than using gene delivery, which is incapable of targeting aspecific cell type and permanently alters a cell's genetic information.An approximately normal cochlear structure is desired with rows of haircells that have supporting cells between them, and hair cells do notcontact other hair cells. Further, it would be desirable to avoid usinggenetic modification to drive proliferation to create large cellaggregations in the cochlea that disrupt the organ's anatomy.

In certain embodiments, the disclosure provides a method for increasingthe cell density of hair cells in an initial population of cochlearcells comprising hair cells and supporting cells. The method comprisesselectively expanding the number of supporting cells in the initialpopulation to form an intermediate cochlear cell population wherein theratio of the number of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of the number ofsupporting cells to hair cells in the initial cochlear cell population.The method further comprises generating hair cells in the intermediatecochlear cell population to form an expanded cochlear cell populationwherein the ratio of the number of hair cells to supporting cells in theexpanded cochlear cell population exceeds the ratio of the number ofhair cells to supporting cells in the intermediate cochlear cellpopulation. In some embodiments, the method does not comprise the use ofa notch activator or an HDAC inhibitor

In certain embodiments, the disclosure provides a method for increasingthe number of Lgr5⁺ supporting cells or increasing the Lgr5⁺ activity inan initial population of cochlear cells, wherein the initial populationcomprises supporting cells and hair cells. For example, in one suchmethod an intermediate population is formed in which the number of Lgr5⁺supporting cells is expanded relative to the initial population.Alternatively, in one such method an intermediate population is formedin which the Lgr5⁺ activity of the supporting cells relative to theinitial population is increased. Alternatively, a method where thenumber of Lgr5⁺ cells is increased relative to the initial cellpopulation by activating Lgr5⁺ expression in cell types that normallylack or have very low levels of Lgr5⁺. In some embodiments, thesealternative methods do not comprise the use of a notch activator or anHDAC inhibitor. By way of further example, an intermediate population isformed in which the number of Lgr5⁺ supporting cells is expanded and theLgr5 activity is increased relative to the initial cochlear cellpopulation. Thereafter, hair cells in the intermediate cochlear cellpopulation may be generated to form an expanded cochlear cell populationwherein the ratio of hair cells to supporting cells in the expandedcochlear cell population exceeds the ratio of the number of hair cellsto supporting cells in the intermediate cochlear cell population.

In each of the aforementioned embodiments of the present disclosure,stemness is induced by activating Wnt or inhibiting GSK3-beta activityor inhibiting GSK-3 alpha activity. In some embodiments, inducingstemness does not comprise the use of a notch activator or an HDACinhibitor

In certain embodiments, the disclosure provides methods for preventingand treating auditory dysfunction. For example, in certain embodiments,the disclosure provides methods for preventing or treating auditoryimpairments in a subject comprising administering to said subject aneffective amount of a compound provided herein.

In certain embodiments, the present disclosure also relates to ex-vivouses of cells described herein. For example, approaches described hereincan be used for discovery purposes. For example, certain embodiments ofthe present disclosure are useful for identifying agents thatproliferate hair cell progenitors and/or increase numbers of hair cells,and also agents that protect supporting cells and/or hair cells (e.g.,to support their survival), and also for identifying agents that aretoxic or not toxic to supporting cells or differentiated progenyincluding hair cells.

In certain embodiments, the disclosure provides for methods forinhibiting the loss or death of the cells of the auditory system in asubject comprising administering to said subject an effective amount ofthe compound described herein or derivative thereof or pharmaceuticallyacceptable salt thereof and an acceptable carrier or excipient, therebyinhibiting loss or death of the cells of the auditory system in thesubject. In some embodiments, the method does not comprise the use of anotch activator or an HDAC inhibitor

In certain embodiments, the disclosure provides methods for maintainingor promoting the growth of cells of the auditory system in a subjectcomprising administering to said subject the compound described hereinor derivative thereof or pharmaceutically acceptable salt thereof in aneffective amount so as to augment or initiate endogenous repair, therebymaintaining or promoting the growth of cells of the auditory system inthe subject.

Also described herein is a method for expanding a population of cochlearcells in a cochlear tissue comprising a parent population of cells, theparent population including supporting cells and a number of Lgr5⁺cells, the method comprising contacting the cochlear tissue with a stemcell proliferator wherein an expanded population of cells is formed inthe cochlear tissue, wherein the stem cell proliferator is capable (i)in a stem cell proliferation assay of increasing the number of Lgr5⁺cells in a stem cell proliferation assay cell population by a factor ofat least 10 and (ii) in a stem cell differentiation assay of forminghair cells from a cell population comprising Lgr5⁺ cells. In someembodiments for expanding a population of cochlear cells, the methoddoes not comprise the use of a notch activator or an HDAC inhibitor

Also described herein is a method for expanding a population of cochlearcells in a cochlear tissue comprising a parent population of cells, theparent population including supporting cells, the method comprisingcontacting the cochlear tissue with a stem cell proliferator to form anexpanded population of cells in the cochlear tissue. The stem cellproliferator can be capable of (i) forming a proliferation assay finalcell population from a proliferation assay initial cell population overa proliferation assay time period in a stem cell proliferation assay and(ii) forming a differentiation assay final cell population from adifferentiation assay initial cell population over a differentiationassay time period in a stem cell differentiation assay wherein: (a) theproliferation assay initial cell population has (i) a proliferationassay initial number of total cells, (ii) a proliferation assay initialnumber of Lgr5⁺ cells, (iii) a proliferation assay initial number ofhair cells, (iv) a proliferation assay initial Lgr5⁺ cell fraction thatequals the ratio of the proliferation assay initial number of Lgr5⁺cells to the proliferation assay initial number of total cells, and (v)a proliferation assay initial hair cell fraction that equals the ratioof the proliferation assay initial number of hair cells to theproliferation assay initial number of total cells; (b) the proliferationassay final cell population has (i) a proliferation assay final numberof total cells, (ii) a proliferation assay final number of Lgr5⁺ cells,(iii) a proliferation assay final number of hair cells, (iv) aproliferation assay final Lgr5⁺ cell fraction that equals the ratio ofthe proliferation assay final number of Lgr5⁺ cells to the proliferationassay final number of total cells and (v) a proliferation assay finalhair cell fraction that equals the ratio of the proliferation assayfinal number of hair cells to the proliferation assay final number oftotal cells; (c) the differentiation assay initial cell population has(i) a differentiation assay initial number of total cells, (ii) adifferentiation assay initial number of Lgr5⁺ cells, (iii) adifferentiation assay initial number of hair cells, (iv) adifferentiation assay initial Lgr5⁺ cell fraction that equals the ratioof the differentiation assay initial number of Lgr5⁺ cells to thedifferentiation assay initial number of total cells, and (v) adifferentiation assay initial hair cell fraction that equals the ratioof the differentiation assay initial number of hair cells to thedifferentiation assay initial number of total cells; (d) thedifferentiation assay final cell population has (i) a differentiationassay final number of total cells, (ii) a differentiation assay finalnumber of Lgr5⁺ cells, (iii) a differentiation assay final number ofhair cells, (iv) a differentiation assay final Lgr5⁺ cell fraction thatequals the ratio of the differentiation assay final number of Lgr5⁺cells to the differentiation assay final number of total cells, and (v)a differentiation assay final hair cell fraction that equals the ratioof the differentiation assay final number of hair cells to thedifferentiation assay final number of total cells; (e) the proliferationassay final number of Lgr5⁺ cells exceeds the proliferation assayinitial number of Lgr5⁺ cells by a factor of at least 10; and (f) thedifferentiation assay final number of hair cells is a non-zero number.In some embodiments of the assay or method described above, the assay ormethod does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

The proliferation assay final number of Lgr5⁺ cells can be greater thanthe proliferation assay initial number of Lgr5⁺ cells by a factor of atleast 50, or by a factor of at least 100. The expanded population ofcells in the cochlear tissue can include a greater number of hair cellsthan does the parent population. The proliferation assay final Lgr5⁺cell fraction can be greater than the differentiation assay initialLgr5⁺ cell fraction by at least a factor of 2. The differentiation assayfinal hair cell fraction can be greater than the proliferation assayinitial hair cell fraction by at least a factor of 2. The proliferationassay final hair cell fraction can be at least 25% less than theproliferation assay initial hair cell fraction. The proliferation assayfinal Lgr5⁺ cell fraction can be at least 10% greater than proliferationassay initial Lgr5⁺ cell fraction. One of more morphologicalcharacteristics of the cochlear tissue can be maintained. Nativemorphology can be maintained. The stem cell proliferator can bedispersed in a biocompatible matrix, which can be a biocompatible gel orfoam. The cochlear tissue can be an in vivo cochlear tissue or an exvivo cochlear tissue. The method can produce a population of Lgr5⁺ cellsthat are in s-phase. The cochlear tissue can be in a subject, andcontacting the cochlear tissue with the compound can be achieved byadministering the compound trans-tympanically to the subject. Contactingthe cochlear tissue with the compound can result in improved auditoryfunctioning of the subject. In some embodiments of the assay or methoddescribed above, the assay or method does not comprise the use of anotch agonist or an HDAC inhibitor. In some embodiments of the assay ormethod described above, the assay or method comprises the use of a GSK3inhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a compound of Formula I,Formula II, or Formula III.

Also described herein is a method of treating a subject who has, or isat risk of developing, hearing loss. The method can includetrans-tympanically administering to a cochlear tissue of the subjectcompound provided herein.

Also described herein is a method of generating Myo7a+ cochlear cells.The method can include contacting Lgr5+ cochlear cells with a compoundprovided herein, thereby generating an expanded population of Lgr5+cells, thereby generating Myo7a+ cochlear cells.

Other objects and features will be in part apparent and in part pointedout hereinafter.

Definitions

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration” refers to introducing a substance into a subject. Insome embodiments, administration is auricular, intraauricular,intracochlear, intravestibular, or transtympanically, e.g., byinjection. In some embodiments, administration is directly to the innerear, e.g., injection through the round or oval, otic capsule, orvestibular canals. In some embodiments, administration is directly intothe inner ear via a cochlear implant delivery system. In someembodiments, the substance is injected transtympanically to the middleear. In certain embodiments “causing to be administered” refers toadministration of a second component after a first component has alreadybeen administered (e.g., at a different time and/or by a differentactor).

An “antibody” refers to an immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in theexpression or activity of a target gene, protein, or a pathway,respectively. Therefore, an agonist can bind to and activate its cognatereceptor in some fashion, which directly or indirectly brings about thisphysiological effect on the target gene or protein. An agonist can alsoincrease the activity of a pathway through modulating the activity ofpathway components, for example, through inhibiting the activity ofnegative regulators of a pathway. Therefore, a “Wnt agonist” can bedefined as an agent that increases the activity of Wnt pathway, whichcan be measured by increased TCF/LEF-mediated transcription in a cell.Therefore, a “Wnt agonist” can be a true Wnt agonist that binds andactivates a Frizzled receptor family member, including any and all ofthe Wnt family proteins, an inhibitor of intracellular beta-catenindegradation, and activators of TCF/LEF.

An “antagonist” refers to an agent that binds to a receptor, and whichin turn decreases or eliminates binding by other molecules.

“Anti-sense” refers to a nucleic acid sequence, regardless of length,that is complementary to the coding strand or mRNA of a nucleic acidsequence. Antisense RNA can be introduced to an individual cell, tissueor organanoid. An anti-sense nucleic acid can contain a modifiedbackbone, for example, phosphorothioate, phosphorodithioate, or othermodified backbones known in the art, or may contain non-naturalinternucleoside linkages.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence capable of hybridizing with another nucleic acidsequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

“Auricular administration” refers to a method of using a catheter orwick device to administer a compound or composition across the tympanicmembrane to the inner ear of the subject. To facilitate insertion of thewick or catheter, the tympanic membrane may be pierced using a suitablysized syringe or pipette. The devices could also be inserted using anyother methods known to those of skill in the art, e.g., surgicalimplantation of the device. In particular embodiments, the wick orcatheter device may be a stand-alone device, meaning that it is insertedinto the ear of the subject and then the compound or composition iscontrollably released to the inner ear. In other particular embodiments,the wick or catheter device may be attached or coupled to a pump orother device that allows for the administration of additional compoundsor compositions. The pump may be automatically programmed to deliverdosage units or may be controlled by the subject or medicalprofessional.

“Biocompatible Matrix” as used herein is a polymeric carrier that isacceptable for administration to humans for the release of therapeuticagents. A Biocompatible Matrix may be a biocompatible gel or foam.

“Cell Aggregate” as used herein shall mean a body cells in the Organ ofCorti that have proliferated to form a cluster of a given cell type thatis greater than 40 microns in diameter and/or produced a morphology inwhich greater than 3 cell layers reside perpendicular to the basilarmembrane. A “Cell Aggregate” can also refer a process in which celldivision creates a body of cells that cause one or more cell types tobreach the reticular lamina, or the boundary between endolymph andperilymph

“Cell Density” as used herein in connection with a specific cell type isthe mean number of that cell type per area in a RepresentativeMicroscopy Sample. The cell types may include but are not limited toLgr5⁺ cells, hair cells, or supporting cells. The Cell Density may beassessed with a given cell type in a given organ or tissue, includingbut not limited to the cochlea or Organ of Corti. For instance, theLgr5⁺ Cell Density in the Organ of Corti is the Cell Density of Lgr5⁺cells as measured across the Organ of Corti. Typically, supporting cellsand Lgr5⁺ cells will be enumerated by taking cross sections of the Organof Corti. Typically, hair cells will be enumerated by looking down atthe surface of the Organ of Corti, though cross sections may be used insome instances, as described in a Representative Microscopy Sample.Typically, Cell Density of Lgr5⁺ cells will be measured by analyzingwhole mount preparations of the Organ of Corti and counting the numberof Lgr5 cells across a given distance along the surface of theepithelia, as described in a Representative Microscopy Sample. Haircells may be identified by their morphological features such as bundlesor hair cell specific stains (e.g., Myosin VIIa, Prestin, vGlut3,Pou4f3, Espin, conjugated-Phalloidin, PMCA2, Ribeye, Atoh1, etc). Lgr5⁺cells may be identified by specific stains or antibodies (e.g., Lgr5-GFPtransgenic reporter, anti-Lgr5 antibody, etc.)

“Cochlear Concentration” as used herein will be the concentration of agiven agent as measured through sampling cochlear fluid. Unlessotherwise noted, the sample should contain a substantial enough portionof the cochlear fluid so that it is approximately representative of theaverage concentration of the agent in the cochlea. For example, samplesmay be drawn from a vestibular canal, and a series of fluid samplesdrawn in series such that individual samples are comprised of cochlearfluid in specified portions of the cochlea

“Complementary nucleic acid sequence” refers to a nucleic acid sequencecapable of hybridizing with another nucleic acid sequence comprised ofcomplementary nucleotide base pairs.

“Cross-Sectional Cell Density” as used herein in connection with aspecific cell type is the mean number of that cell type per area ofcross section through a tissue in a Representative Microscopy Sample.Cross sections of the Organ of Corti can also be used to determine thenumber of cells in a given plane. Typically, hair cells Cross-sectionalCell Density will be measured by analyzing whole mount preparations ofthe Organ of Corti and counting the number of hair cells across a givendistance in cross sections taken along a portion of the epithelia, asdescribed in a Representative Microscopy Sample. Typically,Cross-sectional Cell Density of Lgr5⁺ cells will be measured byanalyzing whole mount preparations of the Organ of Corti and countingthe number of Lgr5⁺ cells across a given distance in cross sectionstaken along a portion of the epithelia, as described in a RepresentativeMicroscopy Sample. Hair cells may be identified by their morphologicalfeatures such as bundles or hair cell specific stains (suitable stainsinclude e.g., Myosin VIIa, Prestin, vGlut3, Pou4f3,conjugated-Phalloidin, PMCA2, Atoh1, etc.). Lgr5⁺ cells may beidentified by specific stains or antibodies (suitable stains andantibodies include fluorescence in situ hybridization of Lgr5 mRNA,Lgr5-GFP transgenic reporter system, anti-Lgr5 antibodies, etc.).

“Decreasing” refers to decreasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99 or 100%, for example, as compared to the level of reference.

“Decreases” also means decreases by at least 1-fold, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000-fold or more, for example, as compared to the level of areference.

“Differentiation Period” as used herein is the duration of time in whichthere is an Effective Stemness Driver Concentration without an EffectiveDifferentiation Inhibition Concentration.

“Effective Concentration” may be the Effective Stemness DriverConcentration for a Stemness Driver or the Effective DifferentiationInhibition Concentration for a Differentiation Inhibitor.

“Effective Differentiation Inhibition Concentration” is the minimumconcentration of a Differentiation Inhibitor that does not allow morethan a 50% increase in the fraction of the total population of cellsthat are hair cells at the end of the Stem Cell Proliferation Assaycompared to the start of the Stem Cell Proliferation Assay In measuringthe Effective Differentiation Inhibition Concentration, a Hair Cellstain for cells may be used with flow cytometry to quantify hair cellsfor a mouse strain that is not an Atoh1-GFP mouse. Alternatively, andAtoh1-GFP mouse strain may be used.

“Effective Release Rate” (mass/time) as used herein is the EffectiveConcentration (mass/volume)*30 uL/1 hour.

“Effective Stemness Driver Concentration” is the minimum concentrationof a Stemness Driver that induces at least 1.5-fold increase in numberof LGR5⁺ cells in a Stem Cell Proliferation Assay compared to the numberof Lgr5⁺ cells in a Stem Cell Proliferation Assay performed without theStemness Driver and with all other components present at the sameconcentrations.

“Eliminate” means to decrease to a level that is undetectable.

“Engraft” or “engraftment” refers to the process of stem or progenitorcell incorporation into a tissue of interest in vivo through contactwith existing cells of the tissue. “Epithelial progenitor cell” refersto a multipotent cell which has the potential to become restricted tocell lineages resulting in epithelial cells.

“Epithelial stem cell” refers to a multipotent cell which has thepotential to become committed to multiple cell lineages, including celllineages resulting in epithelial cells.

“Expanded population of cells” as used herein means an increase of thetotal number of cells present from the total initial number of cellspresent.

“Fragment” refers to a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

“GSK3beta,” “GSK3β,” and “GSK3B” as used interchangeably herein areacronyms for glycogen synthase kinase 3 beta,

“GSK3beta inhibitor” is a compound or composition that inhibits theactivity of GSK3beta.

“GSK3alpha,” “GSK3α,” and “GSK3A” as used interchangeably herein areacronyms for glycogen synthase kinase 3 alpha.

“GSK3alpha inhibitor” is a compound or composition that inhibits theactivity of GSK3alpha.

“GSK3 inhibitor” is a compound or composition that inhibits the activityof GSKalpha and/or GSK3beta.

“Hybridize” refers to pairing to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

An “inhibitor” refers to an agent that causes a decrease in theexpression or activity of a target gene or protein, respectively. An“antagonist” can be an inhibitor, but is more specifically an agent thatbinds to a receptor, and which in turn decreases or eliminates bindingby other molecules.

As used herein, an “inhibitory nucleic acid” is a double-stranded RNA,RNA interference, miRNA, siRNA, shRNA, or antisense RNA, or a portionthereof, or a mimetic thereof, that when administered to a mammaliancell results in a decrease in the expression of a target gene.Typically, a nucleic acid inhibitor comprises at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. Typically, expression of a target gene is reduced by 10%, 25%,50%, 75%, or even 90-100%.

“In vitro Lgr5 activity” refers to the level of expression or activityof Lgr5 in an in vitro population of cells. It may be measured, forexample, in cells derived from a Lgr5-GFP expressing mouse such as aB6.129P2-Lgr5tml(cre/ERT2)Cle/J mouse (also known asLgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No: 008875)by dissociating cells to single cells, staining with propidium iodide(PI), and analyzing the cells using a flow cytometer for Lgr5-GFPexpression. Inner ear epithelial cells from wild-type (non-Lgr5-GFP)mice that passing the same culturing and analyzing procedures can beused as a negative control. Typically, two populations of cells areshown in the bivariate plot with GFP/FITC as one variable, which includeboth GFP positive and GFP negative populations. Lgr5-positive cells areidentified by gating GFP positive cell population. The percentage ofLgr5-positive cells is measured by gating GFP positive cell populationagainst both GFP negative population and the negative control. Thenumber of Lgr5-positive cells is calculated by multiplying the totalnumber of cells by the percentage of Lgr5-positive cells. For cellsderived from non-Lgr5-GFP mice, Lgr5 activity can be measured using ananti-Lgr5 antibody or quantitative-PCR on the Lgr5 gene.

“In vivo Lgr5 activity” as used herein is the level of expression oractivity of Lgr5 in a subject. It may be measured, for example, byremoving an animal's inner ear and measuring Lgr5 protein or Lgr5 mRNA.Lgr5 protein production can be measured using an anti-Lgr5 antibody tomeasure fluorescence intensity as determined by imaging cochlearsamples, where fluorescence intensity is used as a measure of Lgr5presence. Western blots can be used with an anti-Lgr5 antibody, wherecells can be harvested from the treated organ to determine increases inLgr5 protein. Quantitative-PCR or RNA in situ hybridization can be usedto measure relative changes in Lgr5 mRNA production, where cells can beharvested from the inner ear to determine changes in Lgr5 mRNA.Alternatively, Lgr5 expression can be measured using an Lgr5 promoterdriven GFP reporter transgenic system, where the presence or intensityGFP fluoresce can be directly detected using flow cytometry, imaging, orindirectly using an anti-GFP antibody.

“Increase” or “Increases” also means increases by at least 1-fold, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500, 1000-fold or more, for example, as compared to thelevel of a as compared to the level of a reference standard.

“Increasing” refers to increasing by at least 5%, for example, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 99, 100% or more, for example, as compared to the level of areference.

“Intraauricular administration” refers to administration of a compoundor composition to the middle or inner ear of a subject by directlyinjecting the compound or composition.

“Intracochlear” administration refers to direct injection of a compoundor composition across the tympanic membrane and across the round or ovalmembrane into the cochlea.

“Intravestibular” administration refers to direct injection of acompound or composition across the tympanic membrane and across theround or oval membrane into the vestibular organs.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings.

“Lgr5” is an acronym for the Leucine-rich repeat-containing G-proteincoupled receptor 5, also known as G-protein coupled receptor 49 (GPR49)or G-protein coupled receptor 67 (GPR67). It is a protein that in humansis encoded by the Lgr5 gene.

“Lgr5 activity” is defined as the level of activity of Lgr5 in apopulation of cells. In an in vitro cell population, Lgr5 activity maybe measured in an in vitro Lgr5 Activity assay. In an in vivo cellpopulation, Lgr5 activity may be measured in an in vivo Lgr5 Activityassay.

“Lgr5⁺ cell” or “Lgr5-positive cell” as used herein is a cell thatexpresses Lgr5. “Lgr5-cell” as used herein is a cell that is not Lgr5⁺.

“Lineage Tracing” as used herein is using a mouse line that enables fatetracing of any cell that expresses a target gene at the time of reporterinduction. This can include hair cell or supporting cells genes (Sox2,Lgr5, MyosinVIIa, Pou4f3, etc). For example, lineage tracing may use anLgr5-EGFP-IRES-creERT2 mouse crossed with a reporter mouse, which uponinduction, allows one to trace the fate of cells that expressed Lgr5 atthe time of induction. By further example, Lgr5 cells can be isolatedinto single cells and cultured in a Stem Cell Proliferation Assay togenerate colonies, then subsequently differentiated in a DifferentiationAssay and analyzed for cell fate by staining for hair cell and/orsupporting cell proteins and determining the reporter colocalizationwith either hair cell or supporting cell staining to determine the Lgr5cells' fate. In addition, lineage tracing can be performed in cochlearexplants to track supporting cell or hair cell fate within the intactorgan after treatment. For example, Lgr5 cell fate can be determined byisolating the cochlea from a Lgr5-EGFP-IRES-creERT2 mouse crossed with areporter mouse, and inducing the reporter in Lgr5 cells before or duringtreatment. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determining the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. In addition, lineage tracing can beperformed in vivo track supporting cell or hair cell fate within theintact organ after treatment. For example, Lgr5 cell fate can bedetermined inducing a reporter in an Lgr5-EGFP-IRES-creERT2 mousecrossed with a reporter mouse, treating the animal, then isolating thecochlea. The organ can then be analyzed for cell fate by staining forhair cell and/or supporting cell proteins and determining the reportercolocalization with either hair cell or supporting cell staining todetermine the Lgr5 cells' fate. Lineage tracing may be performed usingalternative reporters of interest as is standard in the art.

“Mammal” refers to any mammal including but not limited to human, mouse,rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

“Mean Release Time” as used herein is the time in which one-half of anagent is released into phosphate buffered saline from a carrier in aRelease Assay.

“Native Morphology” as used herein is means that tissue organizationlargely reflects the organization in a healthy tissue.

“Non-human mammal”, as used herein, refers to any mammal that is not ahuman.

As used in relevant context herein, the term “number” of cells can be 0,1, or more cells.

“Organ of Corti” as used herein refers to the sensory cells (inner andouter hair cells) of the hearing organ located in the cochlea.

“Organoid” or “epithelial organoid” refers to a cell cluster oraggregate that resembles an organ, or part of an organ, and possessescell types relevant to that particular organ.

“Population” of cells refers to any number of cells greater than 1, butis preferably at least 1×10³ cells, at least 1×10⁴ cells, at least atleast 1×10⁵ cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least1×10⁸ cells, at least 1×10⁹ cells, or at least 1×10¹⁰ cells.

“Progenitor cell” as used herein refers to a cell that, like a stemcell, has the tendency to differentiate into a specific type of cell,but is already more specific than a stem cell and is pushed todifferentiate into its “target” cell.

“Reference” means a standard or control condition (e.g., untreated witha test agent or combination of test agents).

“Release Assay” as used herein is a test in which the rate of release ofan agent from a Biocompatible Matrix through dialysis membrane to asaline environment. An exemplary Release Assay may be performed byplacing 30 microliters of a compound or composition in 1 ml PhosphateBuffered Saline inside saline dialysis bag with a suitable cutoff, andplacing the dialysis bag within 10 mL of Phosphate Buffered Saline at37° C. The dialysis membrane size may be chosen based on agent size inorder to allow the agent being assessed to exit the membrane. For smallmolecule release, a 3.5-5 kDa cutoff may be used. The Release Rate for acompound or composition may change over time and may be measured in 1hour increments.

“Representative Microscopy Sample” as used herein describes a sufficientnumber of fields of view within a cell culture system, a portion ofextracted tissue, or an entire extracted organ that the average featuresize or number being measured can reasonably be said to represent theaverage feature size or number if all relevant fields were measured. Forexample, in order to assess the hair cell counts at a frequency range onthe Organ of Corti, ImageJ software (NIH) can used to measure the totallength of cochlear whole mounts and the length of individual countedsegments. The total number of inner hair cells, outer hair cells, andsupporting cells can be counted in the entire or fraction of any of thefour cochlear segments of 1200-1400 μm (apical, mid-apical, mid-basal,and basal) at least 3 fields of view at 100 μm field size would bereasonably considered a Representative Microscopy Sample. ARepresentative Microscopy sample can include measurements within a fieldof view, which can be measured as cells per a given distance. ARepresentative Microscopy sample can be used to assess morphology, suchas cell-cell contacts, cochlear architecture, and cellular components(e.g., bundles, synapses).

“Rosette Patterning” is a characteristic cell arrangement in the cochleain which <5% hair cells are adjacent to other hair cells.

The term “sample” refers to a volume or mass obtained, provided, and/orsubjected to analysis. In some embodiments, a sample is or comprises atissue sample, cell sample, a fluid sample, and the like. In someembodiments, a sample is taken from (or is) a subject (e.g., a human oranimal subject). In some embodiments, a tissue sample is or comprisesbrain, hair (including roots), buccal swabs, blood, saliva, semen,muscle, or from any internal organs, or cancer, precancerous, or tumorcells associated with any one of these. A fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. A body tissue can include, but is not limited to, brain, skin,muscle, endometrial, uterine, and cervical tissue or cancer,precancerous, or tumor cells associated with any one of these. In anembodiment, a body tissue is brain tissue or a brain tumor or cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a “sample” is a “primary sample” in that it is obtainedfrom a source (e.g., a subject); in some embodiments, a “sample” is theresult of processing of a primary sample, for example to remove certainpotentially contaminating components and/or to isolate or purify certaincomponents of interest.

“Self-renewal” refers to the process by which a stem cell divides togenerate one (asymmetric division) or two (symmetric division) daughtercells with development potentials that are indistinguishable from thoseof the mother cell. Self-renewal involves both proliferation and themaintenance of an undifferentiated state.

“siRNA” refers to a double stranded RNA. Optimally, an siRNA is 18, 19,20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang atits 3′ end. These dsRNAs can be introduced to an individual cell orculture system. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

“Stem cell” refers to a multipotent cell having the capacity toself-renew and to differentiate into multiple cell lineages.

“Stem Cell Differentiation Assay” as used herein is an assay todetermine the differentiation capacity of stem cells. In an exemplaryStem Cell Differentiation Assay, the number of cells for an initial cellpopulation is harvested from a Atoh1-GFP mouse between the age of 3 to 7days, by isolating the Organ of Corti sensory epithelium, dissociatingthe epithelium into single cells, and passing the cells through a 40 umcell strainer. Approximately 5000 cells are entrapped in 40 μl ofculture substrate (for example: Matrigel (Corning, Growth FactorReduced)) and placed at the center of wells in a 24-well plate with 500μl of an appropriate culture media, growth factors and agent beingtested. Appropriate culture media and growth factors include AdvancedDMEM/F12 with media Supplements (1× N2, 1× B27, 2 mM Glutamax, 10 mMHEPES, 1 mM N-acetylcysteine, and 100 U/ml Penicillin/100 μg/mlStreptomycin) and growth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50ng/ml IGF-1) as well as the agent(s) being assessed are added into eachwell. Cells are cultured for 10 days in a standard cell cultureincubator at 37° C. and 5% CO₂, with media change every 2 days. Thesecells are then cultured by removing the Stem Cell Proliferation Assayagents and replacing with Basal culture media and molecules to drivedifferentiation. An appropriate Basal culture media is Advanced DMEM/F12supplemented with 1× N2, 1× B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin andappropriate molecules to drive differentiation are 3 μM CHIR99021 and 5μM DAPT for 10 days, with media change every 2 days. The number of haircells in a population may be measured by using flow cytometry for GFP.Hair cell differentiation level can further be assessed using qPCR tomeasure hair cell marker (e.g., Myo7a) expression level normalized usingsuitable and unregulated references or housekeeping genes (e.g., Hprt).Hair cell differentiation level can also be assessed by immunostainingfor hair cell markers (e.g., Myosin7a, vGlut3, Espin, PMCAs, Ribeye,conjugated-phalloidin, Atoh1, Pou4f3, etc). Hair cell differentiationlevel can also be assessed by Western Blot for Myosin7a, vGlut3, Espin,PMCAs, Prestin, Ribeye, Atoh1, and Pou4f3.

“Stem Cell Assay” as used herein is an assay in which a cell or a cellpopulation are tested for a series of criteria to determine whether thecell or cell population are stem cells or enriched in stem cells or stemcell markers. In a stem cell assay, the cell/cell population is testedfor stem cell characteristics such as expression of Stem Cell Markers,and further optionally is tested for stem cell function, including thecapacity of self-renewal and differentiation.

“Stem Cell Proliferator” as used herein is a compound that induces anincrease in a population of cells which have the capacity forself-renewal and differentiation.

“Stem Cell Proliferation Assay” as used herein is an assay to determinethe capacity for agent(s) to induce the creation of stem cells from astarting cell population. In an exemplary Stem Cell Proliferation Assay,the number of cells for an initial cell population is harvested from aLgr5-GFP mouse such as a B6.129P2-Lgr5tml(cre/ERT2)Cle/J mouse (alsoknown as Lgr5-EGFP-IRES-creERT2 or Lgr5-GFP mouse, Jackson Lab Stock No:008875) between the age of 3 to 7 days, by isolating the Organ of Cortisensory epithelium and dissociating the epithelium into single cells.Approximately 5000 cells are entrapped in 40 μl of culture substrate(for example: Matrigel (Corning, Growth Factor Reduced)) and placed atthe center of wells in a 24-well plate with 500 μl of an appropriateculture media, growth factors and agent being tested. Appropriateculture media and growth factors include Advanced DMEM/F12 with mediaSupplements (1× N2, 1× B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin) andgrowth factors (50 ng/ml EGF, 50 ng/ml bFGF, and 50 ng/ml IGF-1) as wellas the agent(s) being assessed are added into each well. Cells arecultured for 10 days in a standard cell culture incubator at 37° C. and5% CO₂, with media change every 2 days. The number of Lgr5⁺ cells isquantified by counting the number of cells identified as Lgr5+ in an Invitro Lgr5 activity assay. The fraction of cells that are Lgr5⁺ isquantified by dividing the number of cells identified as Lgr5⁺ in a cellpopulation by the total number of cells present in the cell population.The average Lgr5⁺ activity of a population is quantified by measuringthe average mRNA expression level of Lgr5 of the population normalizedusing suitable and unregulated references or housekeeping genes (e.g.,Hprt). The number of hair cells in a population may be measured bystaining with hair cell marker (e.g., Myosin VIIa), or using anendogenous reporter of hair cell genes (e.g., Pou4f3-GFP, Atoh1-nGFP)and analyzing using flow cytometry. The fraction of cells that are haircells is quantified by dividing the number of cells identified as haircells in a cell population by the total number of cells present in thecell population. Lgr5 activity can be measured by qPCR.

“Stem Cell Markers” as used herein can be defined as gene products(e.g., protein, RNA, etc) that specifically expressed in stem cells. Onetype of stem cell marker is gene products that are directly andspecifically support the maintenance of stem cell identity. Examplesinclude Lgr5 and Sox2. Additional stem cell markers can be identifiedusing assays that have been described in the literature. To determinewhether a gene is required for maintenance of stem cell identity,gain-of-function and loss-of-function studies can be used. Ingain-of-function studies, over expression of specific gene product (thestem cell marker) would help maintain the stem cell identity. While inloss-of-function studies, removal of the stem cell marker would causeloss of the stem cell identity or induced the differentiation of stemcells. Another type of stem cell marker is gene that only expressed instem cells but does not necessary to have specific function to maintainthe identity of stem cells. This type of markers can be identified bycomparing the gene expression signature of sorted stem cells andnon-stem cells by assays such as micro-array and qPCR. This type of stemcell marker can be found in the literature. (e.g., Liu Q. et al., Int JBiochem Cell Biol. 2015 March; 60:99-111.http://www.ncbi.nlm.nih.gov/pubmed/25582750). Potential stem cellmarkers include Ccdc121, Gdf10, Opcm1, Phex, etc. The expression of stemcell markers such as Lgr5 or Sox2 in a given cell or cell population canbe measure using assays such as qPCR, immunohistochemistry, westernblot, and RNA hybridization. The expression of stem cell markers canalso be measured using transgenic cells express reporters which canindicate the expression of the given stem cell markers, e.g., Lgr5-GFPor Sox2-GFP. Flow cytometry analysis can then be used to measure theactivity of reporter expression. Fluorescence microscopy can also beused to directly visualize the expression of reporters. The expressionof stem cell markers may further be determined using microarray analysisfor global gene expression profile analysis. The gene expression profileof a given cell population or purified cell population can be comparedwith the gene expression profile of the stem cell to determinesimilarity between the 2 cell populations. Stem cell function can bemeasured by colony forming assay or sphere forming assay, self-renewalassay and differentiation assay. In colony (or sphere) forming assay,when cultured in appropriate culture media, the stem cell should be ableto form colonies, on cell culture surface (e.g., cell culture dish) orembedded in cell culture substrate (e.g., Matrigel) or be able to formspheres when cultured in suspension. In colony/sphere forming assay,single stem cells are seeded at low cell density in appropriate culturemedia and allowed to proliferate for a given period of time (7-10 days).Colony formed are then counted and scored for stem cell markerexpression as an indicator of stemness of the original cell. Optionally,the colonies that formed are then picked and passaged to test itsself-renewal and differentiation potential. In self-renewal assay, whencultured in appropriate culture media, the cells should maintain stemcell marker (e.g., Lgr5) expression over at least one (e.g., 1, 2, 3, 4,5, 10, 20, etc) cell divisions. In a Stem Cell Differentiation Assay,when cultured in appropriate differentiation media, the cells should beable to generate hair cell which can be identified by hair cell markerexpression measured by qPCR, immunostaining, western blot, RNAhybridization or flow cytometry.

“Stemness Driver” as used herein is a compound or composition thatinduces proliferation of LGR5⁺ cells, upregulates Lgr5 in cells, ormaintains Lgr5 expression in cells, while maintaining the potential forself-renewal and the potential to differentiate into hair cells.Generally, stemness drivers upregulate at least one biomarker ofpost-natal stem cells. Stemness Drivers include but are not limited toWnt agonists, GSK3alpha inhibitors, and GSK3Beta inhibitors.

“Subject” includes humans and mammals (e.g., mice, rats, pigs, cats,dogs, and horses). In many embodiments, subjects are be mammals,particularly primates, especially humans. In some embodiments, subjectsare livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. In someembodiments (e.g., particularly in research contexts) subject mammalswill be, for example, rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like.

“Supporting Cell” as used herein in connection with a cochlearepithelium comprises epithelial cells within the organ of Corti that arenot hair cells. This includes inner pillar cells, outer pillar cells,inner phalangeal cells, Deiter cells, Hensen cells, Boettcher cells,and/or Claudius cells.

“Synergy” or “synergistic effect” is an effect which is greater than thesum of each of the effects taken separately; a greater than additiveeffect.

“TgfBeta inhibitor” as used herein is a compound or composition thatreduces activity of TgfBeta

“Tissue” is an ensemble of similar cells from the same origin thattogether carry out a specific function including, for example, tissue ofcochlear, such as the Organ of Corti.

“Transtympanic” administration refers to direct injection of a compoundor composition across the tympanic membrane into the middle ear.

“Treating” as used herein in connection with a cell population meansdelivering a substance to the population to effect an outcome. In thecase of in vitro populations, the substance may be directly (or evenindirectly) delivered to the population. In the case of in vivopopulations, the substance may be delivered by administration to thehost subject.

“Wnt activation” as used herein is an activation of the Wnt signalingpathway.

The use of “or” means “and/or” unless stated otherwise. As used in thisapplication, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps. As used in this application,the terms “about” and “approximately” are used as equivalents. Anynumerals used in this application with or without about/approximatelyare meant to cover any normal fluctuations appreciated by one ofordinary skill in the relevant art. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid,/toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.For example, inorganic salts include, but are not limited to, ammonium,sodium, potassium, calcium, and magnesium salts. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as ammonia, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Non-limiting examples of organic bases used in certain embodimentsinclude isopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline and caffeine.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compounds or compositions described herein can be formulated in anymanner suitable for a desired delivery route, e.g., transtympanicinjection, transtympanic wicks and catheters, and injectable depots.Typically, formulations include all physiologically acceptablecompositions including derivatives or prodrugs, solvates, stereoisomers,racemates, or tautomers thereof with any physiologically acceptablecarriers, diluents, and/or excipients.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present disclosure relates to methods to activate the Wnt pathway orinhibiting GSK3-beta activity or inhibiting GSK3-alpha activity.Although there are hundreds of purported GSK3 inhibitors in the patentand non patent literature, not all GSK3 inhibitors when administered inthe absence of other therapeutic agents would be sufficient or potentenough to promote activation of stem cell proliferation.

In another aspect the present disclosure relates to methods to prevent,reduce or treat the incidence and/or severity of disorders or diseasesassociated with absence or lack of certain tissue cells. In one aspectthe present disclosure relates to methods to prevent, reduce or treatthe incidence and/or severity of inner ear disorders and hearingimpairments involving inner ear tissue, particularly inner ear haircells, their progenitors, and optionally, the stria vascularis, andassociated auditory nerves. Of particular interest are those conditionsthat lead to permanent hearing loss where reduced number of hair cellsmay be responsible and/or decreased hair cell function. Also of interestare those arising as an unwanted side-effect of ototoxic therapeuticdrugs including cisplatin and its analogs, aminoglycoside antibiotics,salicylate and its analogs, or loop diuretics. In certain embodiments,the present disclosure relates to inducing, promoting, or enhancing thegrowth, proliferation or regeneration of inner ear tissue, particularlyinner ear supporting cells and hair cells.

Among other things, the methods presented here are useful for thepreparation of pharmaceutical formulations for the prophylaxis and/ortreatment of acute and chronic ear disease and hearing loss, dizzinessand balance problems especially of sudden hearing loss, acoustic trauma,hearing loss due to chronic noise exposure, presbycusis, trauma duringimplantation of the inner ear prosthesis (insertion trauma), dizzinessdue to diseases of the inner ear area, dizziness related and/or as asymptom of Meniere's disease, vertigo related and/or as a symptom ofMeniere's disease, tinnitus, and hearing loss due to antibiotics andcytostatics and other drugs.

When cochlea supporting cell populations are treated with the compound,whether the population is in vivo or in vitro, the treated supportingcells exhibit stem-like behavior in that the treated supporting cellshave the capacity to proliferate and differentiate and, morespecifically, differentiate into cochlear hair cells. Preferably, thecompound induces and maintains the supporting cells to produce daughterstem cells that can divide for many generations and maintain the abilityto have a high proportion of the resulting cells differentiate into haircells. In certain embodiments, the proliferating stem cells express stemcell markers which may include Lgr5, Sox2, Opem1, Phex, lin28, Lgr6,cyclin D1, Msx1, Myb, Kit, Gdnf3, Zic3, Dppa3, Dppa4, Dppa5, Nanog,Esrrb, Rex1, Dnmt3a, Dnmt3b, Dnmt31, Utf1, Tel1, Oct4, Klf4, Pax6, Six2,Zic1, Zic2, Otx2, Bmi1, CDX2, STAT3, Smad1, Smad2, smad2/3, smad4,smad5, and/or smad7.

In some embodiments, the method of the present disclosure may be used tomaintain, or even transiently increase stemness (i.e., self-renewal) ofa pre-existing supporting cell population prior to significant hair cellformation. In some embodiments, the pre-existing supporting cellpopulation comprises inner pillar cells, outer pillar cells, innerphalangeal cells, Deiter cells, Hensen cells, Boettcher cells, and/orClaudius cells. Morphological analyses with immunostaining (includingcell counts) and lineage tracing across a Representative MicroscopySamples may be used to confirm expansion of one or more of thesecell-types. In some embodiments, the pre-existing supporting cellscomprise Lgr5⁺ cells. Morphological analyses with immunostaining(including cell counts) and qPCR and RNA hybridization may be used toconfirm Lgr5 upregulation amongst the cell population.

Advantageously, the methods of the present disclosure achieve thesegoals without the use of genetic manipulation. Germ-line manipulationused in many academic studies is not a therapeutically desirableapproach to treating hearing loss. In general, the therapy preferablyinvolves the administration of a small molecule, peptide, antibody, orother non-nucleic acid molecule or nucleic acid delivery vectorunaccompanied by gene therapy. In certain embodiments, the therapyinvolves the administration of a small organic molecule. Preferably,hearing protection or restoration is achieved through the use of a(non-genetic) therapeutic that is injected in the middle ear anddiffuses into the cochlea.

The cochlea relies heavily on all present cell types, and theorganization of these cells is important to their function. Assupporting cells play an important role in neurotransmitter cycling andcochlear mechanics. Thus, maintaining a rosette patterning within theorgan of Corti may be important for function. Cochlear mechanics of thebasilar membrane activate hair cell transduction. Due to the highsensitivity of cochlear mechanics, it is also desirable to avoid massesof cells. In all, maintaining proper distribution and relation of haircells and supporting cells along the basilar membrane, even afterproliferation, is likely a desired feature for hearing as supportingcell function and proper mechanics is necessary for normal hearing.

In one embodiment of the present disclosure, the cell density of haircells in a cochlear cell population is expanded in a manner thatmaintains, or even establishes, the rosette pattern characteristic ofcochlear epithelia.

In accordance with one aspect of the present disclosure, the celldensity of hair cells may be increased in a population of cochlear cellscomprising both hair cells and supporting cells. The cochlear cellpopulation may be an in vivo population (i.e., comprised by the cochlearepithelium of a subject) or the cochlear cell population may be an invitro (ex vivo) population. If the population is an in vitro population,the increase in cell density may be determined by reference to aRepresentative Microscopy Sample of the population taken prior andsubsequent to any treatment. If the population is an in vivo population,the increase in cell density may be determined indirectly by determiningan effect upon the hearing of the subject with an increase in hair celldensity correlating to an improvement in hearing.

In one embodiment, supporting cells placed in a Stem Cell ProliferationAssay in the absence of neuronal cells form ribbon synapses.

In a native cochlea, patterning of hair cells and supporting cellsoccurs in a manner parallel to the basilar membrane. In one embodimentof the present disclosure, the proliferation of supporting cells in acochlear cell population is expanded in a manner that the basilarmembrane characteristic of cochlear epithelia.

In one embodiment, the number of supporting cells in an initial cochlearcell population is selectively expanded by treating the initial cochlearcell population with a compound or composition provided herein to forman intermediate cochlear cell population and wherein the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population. The expanded cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population. For example, in one such embodimentthe ratio of supporting cells to hair cells in the intermediate cochlearcell population exceeds the ratio of supporting cells to hair cells inthe initial cochlear cell population by a factor of 1.1. By way offurther example, in one such embodiment the ratio of supporting cells tohair cells in the intermediate cochlear cell population exceeds theratio of supporting cells to hair cells in the initial cochlear cellpopulation by a factor of 1.5. By way of further example, in one suchembodiment the ratio of supporting cells to hair cells in theintermediate cochlear cell population exceeds the ratio of supportingcells to hair cells in the initial cochlear cell population by a factorof 2. By way of further example, in one such embodiment the ratio ofsupporting cells to hair cells in the intermediate cochlear cellpopulation exceeds the ratio of supporting cells to hair cells in theinitial cochlear cell population by a factor of 3. In each of theforegoing embodiments, the capacity of a compound or composition of thepresent disclosure to expand a cochlear cell population as described inthis paragraph may be determined by means of a Stem Cell ProliferationAssay. In some embodiments of the assay or method described above, theassay or method does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

In one embodiment, the number of stem cells in a cochlear cellpopulation is expanded to form an intermediate cochlear cell populationby treating a cochlear cell population with a compound or compositionprovided herein wherein the cell density of stem cells in theintermediate cochlear cell population exceeds the cell density of stemcells in the initial cochlear cell population. The treated cochlear cellpopulation may be, for example, an in vivo population, an in vitropopulation or even an in vitro explant. In one such embodiment, the celldensity of stem cells in the treated cochlear cell population exceedsthe cell density of stem cells in the initial cochlear cell populationby a factor of at least 1.1. For example, in one such embodiment thecell density of stem cells in the treated cochlear cell populationexceeds the cell density of stem cells in the initial cochlear cellpopulation by a factor of at least 1.25. For example, in one suchembodiment the cell density of stem cells in the treated cochlear cellpopulation exceeds the cell density of stem cells in the initialcochlear cell population by a factor of at least 1.5. By way of furtherexample, in one such embodiment the cell density of stem cells in thetreated cochlear cell population exceeds the cell density of stem cellsin the initial cochlear cell population by a factor of at least 2. Byway of further example, in one such embodiment the cell density of stemcells in the treated cochlear cell population exceeds the cell densityof stem cells in the initial cochlear cell population by a factor of atleast 3. In vitro cochlear cell populations may expand significantlymore than in vivo populations; for example, in certain embodiments thecell density of stem cells in an expanded in vitro population of stemcells may be at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2,000 or even3,000 times greater than the cell density of the stem cells in theinitial cochlear cell population. In each of the foregoing embodiments,the capacity of a compound or composition of the present disclosure toexpand a cochlear cell population as described in this paragraph may bedetermined by means of a Stem Cell Proliferation Assay. In someembodiments of the assay or method described above, the assay or methoddoes not comprise the use of a notch agonist or an HDAC inhibitor. Insome embodiments of the assay or method described above, the assay ormethod comprises the use of a GSK3 inhibitor. In some embodiments of theassay or method described above, the assay or method comprises the useof a compound of Formula I, Formula II, or Formula III.

In accordance with one aspect of the present disclosure, a cochleasupporting cell population is treated with a compound or compositionprovided herein to increase the Lgr5 activity of the population. Forexample, in one embodiment the compound or composition provided hereinhave the capacity to increase and maintain the Lgr5 activity of an invitro population of cochlea supporting cells by factor of at least 1.2.By way of further example, in one such embodiment the compound has thecapacity to increase the Lgr5 activity of an in vitro population ofcochlea supporting cells by factor of 1.5. By way of further example, inone such embodiment the compound has the capacity to increase the Lgr5activity of an in vitro population of cochlea supporting cells by factorof 2, 3, 5 10, 100, 500, 1,000, 2,000 or even 3,000. Increases in Lgr5activity may also be observed for in vivo populations but the observedincrease may be somewhat more modest. For example, in one embodiment thecompound has the capacity to increase the Lgr5 activity of an in vivopopulation of cochlea supporting cells by at least 5%. By way of furtherexample, in one such embodiment the compound has the capacity toincrease the Lgr5 activity of an in vivo population of cochleasupporting cells by at least 10%. By way of further example, in one suchembodiment the compound has the capacity to increase the Lgr5 activityof an in vivo population of cochlea supporting cells by at least 20%. Byway of further example, in one such embodiment the compound has thecapacity to increase the Lgr5 activity of an in vivo population ofcochlea supporting cells by at least 30%. In each of the foregoingembodiments, the capacity of the compound for such an increase in Lgr5activity may be demonstrated, for example, in an In vitro Lgr5⁺ ActivityAssay and in an in vivo population may be demonstrated, for example, inan In vivo Lgr5⁺ Activity Assay, as measured by isolating the organ andperforming morphological analyses using immunostaining, endogenousfluorescent protein expression of Lgr5 (e.g., Lgr5, Sox2), and qPCR forLgr5. In some embodiments of the assay or method described above, theassay or method does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

In addition to increasing the Lgr5 activity of the population, thenumber of Lgr5⁺ supporting cells in a cochlea cell population may beincreased by treating a cochlea cell population containing Lgr5⁺supporting cells (whether in vivo or in vitro) with a compound orcomposition provided herein. In general, the cell density of thestem/progenitor supporting cells may expand relative to the initial cellpopulation via one or more of several mechanisms. For example, in onesuch embodiment, newly generated Lgr5⁺ supporting cells may be generatedthat have increased stem cell propensity (i.e., greater capacity todifferentiate into hair cell). By way of further example, in one suchembodiment no daughter Lgr5⁺ cells are generated by cell division, butpre-existing Lgr5⁺ supporting cells are induced to differentiate intohair cells. By way of further example, in one such embodiment nodaughter cells are generated by cell division, but Lgr5⁻ supportingcells are activated to a greater level of Lgr5 activity and theactivated supporting cells are then able to differentiate into haircells. Regardless of the mechanism, in one embodiment the compound ofthe present disclosure has the capacity to increase the cell density ofLgr5⁺ supporting cells in an in vitro isolated cell population ofcochlea supporting cells by factor of at least 5. By way of furtherexample, in one such embodiment the compound has the capacity toincrease the cell density of Lgr5⁺ supporting cells in an in vitropopulation of cochlea supporting cells by factor of at least 10. By wayof further example, in one such embodiment the compound has the capacityto increase the cell density of Lgr5⁺ supporting cells in an in vitropopulation of cochlea supporting cells by factor of at least 100, atleast 500, at least 1,000 or even at least 2,000. Increases in the celldensity of Lgr5⁺ supporting cells may also be observed for in vivopopulations but the observed increase may be somewhat more modest. Forexample, in one embodiment the compound has the capacity to increase thecell density of Lgr5⁺ supporting cells in an in vivo population ofcochlea supporting cells by at least 5%. By way of further example, inone such embodiment the compound has the capacity to increase the celldensity of Lgr5⁺ supporting cells in an in vivo population of cochleasupporting cells by at least 10%. By way of further example, in one suchembodiment the compound has the capacity to increase the cell density ofLgr5⁺ supporting cells in an in vivo population of cochlea supportingcells by at least 20%. By way of further example, in one such embodimentthe compound has the capacity to increase the cell density of Lgr5⁺supporting cells in an in vivo population of cochlea supporting cells byat least 30%. The capacity of the compound for such an increase in Lgr5⁺supporting cells in an in vitro population may be demonstrated, forexample, in a Stem Cell Proliferation Assay or in an appropriate in vivoassay. In one embodiment, a compound of the present disclosure has thecapacity to increase the number of Lgr5⁺ cells in the cochlea byinducing expression of Lgr5 in cells with absent or low detection levelsof the protein, while maintaining Native Morphology. In one embodiment,a compound of the present disclosure has the capacity to increase thenumber of Lgr5⁺ cells in the cochlea by inducing expression of Lgr5 incells with absent or low detection levels of the protein, whilemaintaining Native Morphology and without producing Cell Aggregates. Insome embodiments of the assay or method described above, the assay ormethod does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

In addition to increasing the cell density of Lgr5⁺ supporting cells, inone embodiment the method of the present disclosure has the capacity toincrease the ratio of Lgr5⁺ cells to hair cells in a cochlear cellpopulation. In one embodiment, the number of Lgr5⁺ supporting cells inan initial cochlear cell population is selectively expanded by treatingthe initial cochlear cell population with a compound of the presentdisclosure to form an expanded cell population and wherein the number ofLgr5⁺ supporting cells in the expanded cochlear cell population at leastequals the number of hair cells. The expanded cochlear cell populationmay be, for example, an in vivo population, an in vitro population oreven an in vitro explant. In one such embodiment, the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1:1. For example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 1.5:1. By way of further example, in one such embodiment theratio of Lgr5⁺ supporting cells to hair cells in the expanded cochlearcell population is at least 2:1. By way of further example, in one suchembodiment the ratio of Lgr5⁺ supporting cells to hair cells in theexpanded cochlear cell population is at least 3:1. By way of furtherexample, in one such embodiment the ratio of Lgr5⁺ supporting cells tohair cells in the expanded cochlear cell population is at least 4:1. Byway of further example, in one such embodiment the ratio of Lgr5⁺supporting cells to hair cells in the expanded cochlear cell populationis at least 5:1. In each of the foregoing embodiments, the capacity ofthe compound of the present disclosure to expand a cochlear cellpopulation as described in this paragraph may be determined by means ofa Stem Cell Proliferation Assay. In some embodiments of the assay ormethod described above, the assay or method does not comprise the use ofa notch agonist or an HDAC inhibitor. In some embodiments of the assayor method described above, the assay or method comprises the use of aGSK3 inhibitor. In some embodiments of the assay or method describedabove, the assay or method comprises the use of a compound of Formula I,Formula II, or Formula III.

In certain embodiments, the method increases the fraction of the Lgr5⁺cells to total cells on the sensory epithelium by at least 10%, 20%,50%, 100%, 250% 500%, 1,000% or 5000%.

In certain embodiments, the method increases the Lgr5⁺ cells until theybecome at least 10, 20, 30, 50, 70, or 85% of the cells on the sensoryepithelium, e.g., the Organ of Corti.

In general, excessive proliferation of supporting cells in the cochleais preferably avoided. In one embodiment, the method of the presentdisclosure has the capacity to expand a cochlear cell population withoutcreating a protrusion of new cells beyond the native surface of thecochlea, e.g., a Cell Aggregate. In some embodiments, 30 days afterplacing a compound or composition provided herein on the round or ovalmembrane, the cochlear tissue has Native Morphology. In someembodiments, 30 days after placing the compound on the round or ovalmembrane, the cochlear tissue has Native Morphology and lacks CellAggregates. In some embodiments, 30 days after placing the compound onthe round or oval membrane, the cochlear tissue has Native Morphologyand at least 10, 20, 30, 50, 75, 90, 95, 98, or even at least 99% of theLgr5⁺ cells in the Organ of Corti are not part of Cell Aggregates. Insome embodiments of the assay or method described above, the assay ormethod does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

In addition to expanding supporting cell populations, generally, andLgr5⁺ supporting cells, specifically, as described above, the method ofthe present disclosure has the capacity to maintain, in the daughtercells, the capacity to differentiate into hair cells. In in vivopopulations, the maintenance of this capacity may be indirectly observedby an improvement in a subject's hearing. In in vitro populations, themaintenance of this capacity may be directly observed by an increase inthe number of hair cells relative to a starting population or indirectlyby measuring LGR5 activity, SOX2 activity or one or more of the otherstem cell markers identified elsewhere herein. In some embodiments ofthe assay or method described above, the assay or method does notcomprise the use of a notch agonist or an HDAC inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a GSK3 inhibitor. In some embodiments of the assayor method described above, the assay or method comprises the use of acompound of Formula I, Formula II, or Formula III.

In one embodiment, the capacity of the method to increase the stemnessof a population of cochlear supporting cells, in general, or apopulation of Lgr5⁺ supporting cells, in particular, may be correlatedwith an increase of Lgr5 activity of an in vitro population of isolatedLgr5⁺ cells as determined by an Lgr5 Activity Assay. As previouslynoted, in one such embodiment, the compound has the capacity to increasethe Lgr5 activity of stem cells in the intermediate cell population by afactor of 5 on average relative to the Lgr5 activity of the cells in theinitial cell population. By way of further example, in one suchembodiment the method has the capacity to increase the Lgr5 activity ofthe stem cells genes in the intermediate cell population by a factor of10 relative to the Lgr5 activity of the cells in the initial cellpopulation. By way of further example, in one such embodiment the methodhas the capacity to increase the Lgr5 activity of the stem cells in theintermediate cell population by a factor of 100 relative to the Lgr5activity of the cells in the initial cell population. By way of furtherexample, in one such embodiment the method has the capacity to increasethe Lgr5 activity of the stem cells in the intermediate cell populationby a factor of 1000 relative to the Lgr5 activity of the cells in theinitial cell population. In each of the foregoing embodiments, theincrease in the activity of stem cells in the cell population may bedetermined in vitro by immunostaining or endogenous fluorescent proteinexpression for target genes and analysis of their relative intensitiesvia imaging analysis or flowcytometry, or using qPCR for target stemcell genes. The identity of the resulting stem cell population mayoptionally be further determined by stem cell assays including stem cellmarker expression assay, colony forming assay, self-renewal assay anddifferentiation assay as defined in Stem cell assay. In some embodimentsof the assay or method described above, the assay or method does notcomprise the use of a notch agonist or an HDAC inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a GSK3 inhibitor. In some embodiments of the assayor method described above, the assay or method comprises the use of acompound of Formula I, Formula II, or Formula III.

In some embodiments, the method is applied to an adult mammal produces apopulation of adult mammalian Lgr5⁺ cells that are in S-phase.

In one embodiment, after applying the compound or composition providedherein to the round or oval of a mouse, the in vivo Lgr5⁺ Activity of acell population in the Organ of Corti increases 1.3×, 1.5×, up to 20×over baseline for a population that has not been exposed to thecompound. In some embodiments, applying the compound to the round oroval of a mouse increases the average In vivo Lgr5⁺ Activity for cellsin the Organ of Corti is increased 1.3×, 1.5×, up to 20× over baselinefor a population that has not been exposed to the compound.

In certain embodiments, the method increases the Lgr5⁺ cells until theybecome at least 10%, 7.5%, 10%, up to 100% of the supporting cellpopulation by number.

In certain embodiments, the compound has the capacity to increase thepercentage of Lgr5⁺ cell in a cochlea by 5%, 10%, 25%, 50%, or 80%.

In certain embodiments, the stem cell population is of an in vivosubject, and the method is a treatment for hearing loss and/orvestibular dysfunction (e.g., wherein the generation of inner ear haircells from the expanded population of stem cells results in partial orfull recovery of hearing loss and/or improved vestibular function). Incertain embodiments, the stem cell population is of an in vivo subject,and the method further comprises delivering a drug to the subject (e.g.,for treatment of a disease and/or disorder unrelated to hearing lossand/or vestibular dysfunction) at a higher concentration than a knownsafe maximum dosage of the drug for the subject (e.g., the known safemaximum dosage if delivered in the absence of the generation of innerear hair cells resulting from the method) (e.g., due to a reduction orelimination of a dose-limiting ototoxicity of the drug). In someembodiments of the assay or method described above, the assay or methoddoes not comprise the use of a notch agonist or an HDAC inhibitor. Insome embodiments of the assay or method described above, the assay ormethod comprises the use of a GSK3 inhibitor. In some embodiments of theassay or method described above, the assay or method comprises the useof a compound of Formula I, Formula II, or Formula III.

In certain embodiments, the method further comprises performing highthroughput screening using the generated inner ear hair cells. Incertain embodiments, the method comprises using the generated inner earhair cells to screen molecules for toxicity against inner ear haircells. In certain embodiments, the method comprises using the generatedinner ear hair cells to screen molecules for ability to improve survivalof inner ear hair cells (e.g., inner ear hair cells exposed to saidmolecules).

In another aspect, the disclosure is directed to a method of producingan expanded population of stem cells, the method comprising:administering or causing to be administered to a stem cell population(e.g., of an in vitro, ex vivo, or in vivo sample/subject) a compound orcomposition provided herein. In some embodiments of the assay or methoddescribed above, the assay or method does not comprise the use of anotch agonist or an HDAC inhibitor. In some embodiments of the assay ormethod described above, the assay or method comprises the use of a GSK3inhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a compound of Formula I,Formula II, or Formula III.

In certain embodiments, the administering step is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In certain embodiments, the administering step comprises administeringthe GSK3-beta inhibitor and/or GSK3-alpha inhibitor and/or Wnt agonistin a sustained manner.

In certain embodiments, the stem cells are inner ear stem cells and/orsupporting cells.

In certain embodiments, the method further comprises performing highthroughput screening using the generated expanded population of stemcells. In certain embodiments, the method further comprises using thegenerated stem cells to screen molecules for toxicity against stem cellsand/or their progeny. In certain embodiments, the method comprises usingthe generated stem cells to screen molecules for ability to improvesurvival of stem cells and/or their progeny.

In another aspect, the disclosure is directed to a method of treating asubject who has, or is at risk of developing, hearing loss and/orvestibular dysfunction, the method comprising: identifying a subject whohas experienced, or is at risk for developing, hearing loss and/orvestibular dysfunction, administering or causing to be administered acompound or composition provided herein. In some embodiments of theassay or method described above, the assay or method does not comprisethe use of a notch agonist or an HDAC inhibitor. In some embodiments ofthe assay or method described above, the assay or method comprises theuse of a GSK3 inhibitor. In some embodiments of the assay or methoddescribed above, the assay or method comprises the use of a compound ofFormula I, Formula II, or Formula III.

In certain embodiments, the stem cell population comprises Lgr5⁺ cells.In certain embodiments, the stem cell population comprises post-natalcells. In certain embodiments, the stem cell population comprisesepithelial stem cells. In certain embodiments, stem cells includeprogenitor cells.

In certain embodiments, the step of administering is carried out byperforming one or more injections into the ear (e.g., transtympanicallyinto the middle ear and/or inner ear).

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating stem cells inan initial stem cell population (e.g., of an in vitro, ex vivo, or invivo sample/subject), resulting in an expanded population of stem cells(e.g., such that the expanded population is a factor of at least 1.25,1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation); and facilitating generation of inner ear hair cells fromthe expanded population of stem cells. In some embodiments of the assayor method described above, the assay or method does not comprise the useof a notch agonist or an HDAC inhibitor. In some embodiments of theassay or method described above, the assay or method comprises the useof a GSK3 inhibitor. In some embodiments of the assay or methoddescribed above, the assay or method comprises the use of a compound ofFormula I, Formula II, or Formula III.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising administering a compound orcomposition provided herein to a cell population in an inner ear of asubject, thereby facilitating generation of inner ear hair cells. Insome embodiments of the assay or method described above, the assay ormethod does not comprise the use of a notch agonist or an HDACinhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a GSK3 inhibitor. In someembodiments of the assay or method described above, the assay or methodcomprises the use of a compound of Formula I, Formula II, or FormulaIII.

In another aspect, the disclosure is directed to a method of generatinginner ear hair cells, the method comprising: proliferating post-natalLGR5+ cells in an initial population (e.g., of an in vitro, ex vivo, orin vivo sample/subject), resulting in an expanded population of LGR5+cells (e.g., such that the expanded population is a factor of at least1.25, 1.5, 1.75, 2, 3, 5, 10, or 20 greater than the initial stem cellpopulation), said expanded population of LGR5+ cells resulting ingeneration of inner ear hair cells. In certain embodiments, stem cellsinclude progenitor cells. In some embodiments of the assay or methoddescribed above, the assay or method does not comprise the use of anotch agonist or an HDAC inhibitor. In some embodiments of the assay ormethod described above, the assay or method comprises the use of a GSK3inhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a compound of Formula I,Formula II, or Formula III.

In another aspect, the disclosure is directed to a method of treating adisease or disorder, the method comprising: proliferating post-natalLgr5⁺ epithelial cells in an initial population of a subject (in vivo),resulting in an expanded population of Lgr5+ epithelial cells (e.g.,such that the expanded population is a factor of at least 1.25, 1.5,1.75, 2, 3, 5, 10, or 20 greater than the initial post-natal Lgr5⁺epithelial cell population). In some embodiments of the assay or methoddescribed above, the assay or method does not comprise the use of anotch agonist or an HDAC inhibitor. In some embodiments of the assay ormethod described above, the assay or method comprises the use of a GSK3inhibitor. In some embodiments of the assay or method described above,the assay or method comprises the use of a compound of Formula I,Formula II, or Formula III.

In some embodiments, Lgr5⁺ cells are differentiated into hair cells.

In some embodiments of the methods described herein, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 0.01 uM toabout 1,000 mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is a compound of Formula III used at aconcentration of about 0.1 uM to about 10 mM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 1 uM toabout 1 mM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is a compound of Formula III used at aconcentration of about 1 uM to about 100 uM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 10 uM andoptionally in combination with other agents.

In some embodiments of the methods described herein, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 0.01 nM toabout 1,000 uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is a compound of Formula III used at aconcentration of about 0.1 nM to about 10 uM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 1 nM toabout 1 uM and optionally in combination with other agents. In otherembodiments, the GSK-3 inhibitor is a compound of Formula III used at aconcentration of about 1 nM to about 100 nM and optionally incombination with other agents. In other embodiments, the GSK-3 inhibitoris a compound of Formula III used at a concentration of about 10 nM andoptionally in combination with other agents.

Administration

The membrane of the round or oval is the biological barrier to the innerear space and represents the major obstacle for the local treatment ofhearing impairment. The administered drug must overcome this membrane toreach the inner ear space. The drug can operatively (e.g., injectionthrough the tympanic membrane) be placed locally to the round or ovalmembrane and can then penetrate through the round or oval membrane.Substances that penetrate the round or oval typically distribute in theperilymph and thus reach the hair cells and supporting cells.

In certain embodiments, pharmaceutical formulations are adapted toadminister the drug locally to the round or oval membrane. Thepharmaceutical formulations may also contain a membrane penetrationenhancer, which supports the passage of the agents mentioned hereinthrough the round or oval membrane. Accordingly, liquid, gel or foamformulations may be used. It is also possible to apply the activeingredient orally or to employ a combination of delivery approaches.

Intratympanic (IT) delivery of drugs to the ear is increasingly used forboth clinical and research purposes. Some groups have applied drugs in asustained manner using microcatheters and microwicks, while the majorityhave applied them as single or as repeated IT injections (up to 8injections over periods of up to 2 weeks).

Intratympanically applied drugs are thought to enter the fluids of theinner ear primarily by crossing the round or oval (RW) membrane.Calculations show that a major factor controlling both the amount ofdrug entering the ear and the distribution of drug along the length ofthe ear is the duration the drug remains in the middle ear space.Single, ‘one-shot’ applications or applications of aqueous solutions forfew hours' duration result in steep drug gradients for the appliedsubstance along the length of the cochlea and rapidly decliningconcentration in the basal turn of the cochlea as the drug subsequentlybecomes distributed throughout the ear.

Other injection approaches include by osmotic pump, or, by combinationwith implanted biomaterial, and more preferably, by injection orinfusion. Biomaterials that can aid in controlling release kinetics anddistribution of drug include hydrogel materials, degradable materials.One class of materials that is most preferably used includes in situgelling materials. Other materials include collagen or other naturalmaterials including fibrin, gelatin, and decellularized tissues. Gelfoammay also be suitable.

Delivery may also be enhanced via alternate means including, but notlimited to, agents added to the delivered compound or composition suchas penetration enhancers, or could be through devices via ultrasound,electroporation, or high speed jet.

Methods described herein can also be used for inner ear cell types thatmay be produced using a variety of methods know to those skilled in theart including those cell types described in PCT Application No.WO2012103012 A1.

With regard to human and veterinary treatment, the amount of aparticular agent(s) that is administered may be dependent on a varietyof factors, including the disorder being treated and the severity of thedisorder; activity of the specific agent(s) employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific agent(s) employed; the duration of the treatment; drugs used incombination or coincidental with the specific agent(s) employed; thejudgment of the prescribing physician or veterinarian; and like factorsknown in the medical and veterinary arts.

The agents described herein may be administered in a therapeuticallyeffective amount to a subject in need of treatment. Administration ofcompounds described herein can be via any of suitable route ofadministration, particularly by intratympanically. Other routes includeingestion, or alternatively parenterally, for example intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrastemally, intracranially, intramuscularly,intranasally, subcutaneously, sublingually, transdermally, or byinhalation or insufflations, or topical by ear instillation forabsorption through the skin of the ear canal and membranes of theeardrum. Such administration may be as a single or multiple oral dose,defined number of ear drops, or a bolus injection, multiple injections,or as a short- or long-duration infusion. Implantable devices (e.g.,implantable infusion pumps) may also be employed for the periodicparenteral delivery over time of equivalent or varying dosages of theparticular formulation. For such parenteral administration, thecompounds are preferably formulated as a sterile solution in water oranother suitable solvent or mixture of solvents. The solution maycontain other substances such as salts, sugars (particularly glucose ormannitol), to make the solution isotonic with blood, buffering agentssuch as acetic, citric, and/or phosphoric acids and their sodium salts,and preservatives.

Compounds described herein can be administered by a number of methodssufficient to deliver the compound to the inner ear. Delivering acompound to the inner ear includes administering the compound to themiddle ear, such that the compound may diffuse across the round or ovalto the inner ear and administering a compound to the inner ear by directinjection through the round or oval membrane. Such methods include, butare not limited to auricular administration, by transtympanic wicks orcatheters, or parenteral administration, for example, by intraauricular,transtympanic, or intracochlear injection.

In particular embodiments, the compounds, compositions and formulationsof the disclosure are locally administered, meaning that they are notadministered systemically.

In one embodiment, a syringe and needle apparatus is used to administercompounds or compositions to a subject using auricular administration. Asuitably sized needle is used to pierce the tympanic membrane and a wickor catheter comprising the compound or composition is inserted throughthe pierced tympanic membrane and into the middle ear of the subject.The device may be inserted such that it is in contact with the round oroval or immediately adjacent to the round or oval. Exemplary devicesused for auricular administration include, but are not limited to,transtympanic wicks, transtympanic catheters, round or ovalmicrocatheters (small catheters that deliver medicine to the round oroval), and Silverstein Microwicks™ (small tube with a “wick” through thetube to the round or oval, allowing regulation by subject or medicalprofessional).

In another embodiment, a syringe and needle apparatus is used toadminister compounds or compositions to a subject using transtympanicinjection, injection behind the tympanic membrane into the middle and/orinner ear. The formulation may be administered directly onto the roundor oval membrane via transtympanic injection or may be administereddirectly to the cochlea via intracochlear injection or directly to thevestibular organs via intravestibular injection.

In some embodiments, the delivery device is an apparatus designed foradministration of compounds or compositions to the middle and/or innerear. By way of example only: GYRUS Medical GmbH offers micro-otoscopesfor visualization of and drug delivery to the round or oval niche;Arenberg has described a medical treatment device to deliver fluids toinner ear structures in U.S. Pat. Nos. 5,421,818; 5,474,529; and5,476,446, each of which is incorporated by reference herein for suchdisclosure. U.S. patent application Ser. No. 08/874,208, which isincorporated herein by reference for such disclosure, describes asurgical method for implanting a fluid transfer conduit to delivercompositions to the inner ear. U.S. Patent Application Publication2007/0167918, which is incorporated herein by reference for suchdisclosure, further describes a combined otic aspirator and medicationdispenser for transtympanic fluid sampling and medicament application.

In some embodiments, a compound or composition provided herein isadministered to a subject in need thereof once. In some embodiments, acompound or composition provided herein is administered to a subject inneed thereof more than once. In some embodiments, a first administrationof a compound or composition provided herein is followed by a second,third, fourth, or fifth administration of a compound or compositionprovided herein.

The number of times a compound is administered to an subject in needthereof depends on the discretion of a medical professional, thedisorder, the severity of the disorder, and the subject's response tothe formulation. In some embodiments, the compound disclosed herein isadministered once to a subject in need thereof with a mild acutecondition. In some embodiments, the compound disclosed herein isadministered more than once to a subject in need thereof with a moderateor severe acute condition. In the case wherein the subject's conditiondoes not improve, upon the doctor's discretion the compound may beadministered chronically, that is, for an extended period of time,including throughout the duration of the subject's life in order toameliorate or otherwise control or limit the symptoms of the subject'sdisease or condition.

In the case wherein the subject's status does improve, upon the doctor'sdiscretion the compound may administered continuously; alternatively,the dose of drug being administered may be temporarily reduced ortemporarily suspended for a certain length of time (i.e., a “drugholiday”). The length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dosereduction during a drug holiday may be from 10%-100%, including by wayof example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once the subject's hearing and/or balance has improved, a maintenancedose can be administered, if necessary. Subsequently, the dosage or thefrequency of administration, or both, is optionally reduced, as afunction of the symptoms, to a level at which the improved disease,disorder or condition is retained. In certain embodiments, subjectsrequire intermittent treatment on a long-term basis upon any recurrenceof symptoms.

Examples

Assay: Mouse Strains

Lgr5-EGFP-IRES-Cre-ER mice (Barker et al., 2007) are used to analyze theeffects of small molecules on cochlear stem cell expansion.

Isolation of stem cells from the inner ear: All animal studies areconducted under an approved institutional protocol according to NationalInstitutes of Health guidelines. For experiments with neonatal mice(postnatal days 1-3), the cochleae are dissected in HBSS and the organof Corti are separated from the stria vascularis and the modiolus. Theorgans of Corti are then treated with Cell Recovery Solution (Corning)for 1 h to separate cochlear epithelium from the underlying mesenchyme.Epithelia are then collected and treated with TrypLE (Life Technologies)for 15-20 minutes at 37° C. Single cells obtained by mechanicaltrituration are filtered (40 μm) and suspended in Matrigel (Corning) for3D culture.

Expansion of Lgr5-Positive Cells

Cells are cultured in a 1:1 mixture of DMEM and F12, supplemented withGlutamax (GIBCO), N2, B27 (Invitrogen), EGF (50 ng/mL; Chemicon), bFGF(50 ng/mL; Chemicon), IGF1 (50 ng/mL; Chemicon) and the compound orcomposition provided herein. Media are changed every other day.

Differentiation of Lgr5-Positive Progenitor Cells Stem cell colonies aredifferentiated in a 1:1 mixture of DMEM and F12, supplemented withGlutamax (GIBCO), N2, B27 (Invitrogen), with addition of specific drugsor after removal of growth factors without drug addition. The specificdrugs are added to the culture to test their effect on differentiation.

The specific drugs used in the assay above are agents that help drivedifferentiation. Non-limiting examples of the specific drugs used in theassay above include gamma secretase inhibitors (e.g., DAPT or LY411575),Wnt activators/GSK3 inhibitors (e.g., Wnt3a, R-spondin, CHIR99021, GSK3inhibitor XXII, AZD1080, etc), antibodies, peptides, siRNA, or acombination thereof.

Analysis

Lgr5-positive cells are quantified after 10 days (D10) in culture inmultiple conditions. Cell colonies are dissociated into single cellsusing TrypLE (Gibco). The cells are then stained with propidium iodide(PI) and are analyzed using a flow cytometer for Lgr5-GFP expression.The number of GFP-positive cells and the percentage of GFP-positivecells are quantified.

Atoh1-nGFP-positive cells are quantified at day 0 (D0) and day 10 (D10)of differentiation treatment to determine the number of hair cells thathave differentiated. Cell colonies are incubated in Cell RecoverySolution to release the colonies from Matrigel and dissociated intosingle cells using TrypLE. The total number and percentage ofGFP-positive cells are quantified using a flow cytometer for multipleculture conditions. ANOVA is used to compare means across conditions,and the two-tailed Student's T-test is used to compare each condition tothe treatment with the highest yield.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Suchembodiments are also within the scope of the following claims. Therecitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof. Theteachings of all patents, published applications and references citedherein are incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

The invention claimed is:
 1. A method for expanding a population ofcochlear cells in a cochlear tissue comprising a parent population ofcells, said method comprising contacting the cochlear tissue with a stemcell proliferator wherein the stem cell proliferator is a1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compoundhaving the following structural moiety of Formula I within the compound:

or a pharmaceutically acceptable salt thereof, wherein an expandedpopulation of cells is formed in the cochlear tissue, wherein the stemcell proliferator is capable (i) in a stem cell proliferation assay ofincreasing the number of Lgr5⁺ cells in a stem cell proliferation assaycell population by a factor of at least 10 and (ii) in a stem celldifferentiation assay of forming hair cells from a cell populationcomprising Lgr5⁺ cells, and wherein the method does not comprise a notchactivator or an HDAC inhibitor.
 2. The method of claim 1, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compoundcomprises a3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound, or a pharmaceutically acceptable salt thereof,having the following structural moiety of Formula II within thecompound:


3. The method of claim 2, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione,or a pharmaceutically acceptable salt thereof, having a Formula III:


4. The method of claim 1, wherein the cochlear tissue is in a subject.5. The method of claim 4, wherein the contacting the cochlear tissuewith the compound is achieved by administering the compoundtrans-tympanically to the subject.
 6. The method of claim 1, whereincontacting the cochlear tissue with the compound results in improvedauditory functioning of the subject.
 7. A method of facilitating thegeneration of tissue cells, the method comprising administering orcausing to be administered to a stem cell population a1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compound,or a pharmaceutically acceptable salt thereof, having the followingstructural moiety of Formula I within the compound:

wherein the method does not comprise a notch activator or an HDACinhibitor.
 8. The method of claim 7, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compoundcomprises a3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound, or a pharmaceutically acceptable salt thereof,having the following structural moiety of Formula II within thecompound:


9. The method of claim 7, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione,or a pharmaceutically acceptable salt thereof, having a Formula III:


10. The method of claim 9 wherein the tissue cells are cochlear cells.11. The method of claim 9, wherein the tissue cells are inner ear haircells.
 12. A method of treating a subject who has, or is at risk ofdeveloping, a disease associated with absence or lack of certain tissuecells, the method administering or causing to be administered to saidsubject a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containingcompound, or a pharmaceutically acceptable salt thereof, having thefollowing structural moiety of Formula I within the compound:

wherein the method does not comprise a notch activator or an HDACinhibitor.
 13. The method of claim 12, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compoundcomprises a3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound, or a pharmaceutically acceptable salt thereof,having the following structural moiety of Formula II within thecompound:


14. The method of claim 12, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione,or a pharmaceutically acceptable salt thereof, having a Formula III:


15. The method of claim 12, wherein the tissue cells are cochlear cells.16. The method of claim 12, wherein the tissue cells are inner ear haircells.
 17. A method of treating a subject who has, or is at risk ofdeveloping, hearing loss, the method comprising administering to thesubject a 1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containingcompound, or a pharmaceutically acceptable salt thereof, having thefollowing structural moiety of Formula I within the compound:

wherein the method does not comprise a notch activator or an HDACinhibitor.
 18. The method of claim 17, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7, 1-hi]indolyl containing compoundcomprises a3-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-1H-pyrrole-2,5-dionecontaining compound, or a pharmaceutically acceptable salt thereof,having the following structural moiety of Formula II within thecompound:


19. The method of claim 17, wherein the1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indole containing compound is3-(9-fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione,or a pharmaceutically acceptable salt thereof, having a Formula III:


20. The method of claim 19, wherein the compound is administeredtrans-tympanically to a cochlear tissue of the subject.